Part:BBa_K4849009:Design

PcopM-NuiA-TrrnB

Design and Experimental confirmation

Our intention was to use the NuiA as an inhibitor in the NucA-based kill switch in Synechocystis sp. PCC6803. We performed the Level 1 assembly using BsaI enzyme (Vasudevan et al., 2019; Gale et al., 2019) to ligate Level 0 Parts containing the PcopM195-BCD promoter (BBa_K4849002), the NuiA CDS (BBa_K4849004), and the TrrnB terminator (BBa_K4013002) into a Level 1 Position 1 (L1P1) vector (https://www.addgene.org/browse/sequence/69000/). This Level 1 NuiA transcriptional unit was later used for Level T assembly into a Level T acceptor vector together with Level 1 NucA transcriptional unit for construction of the kill switch. The assembled L1P1-PcopM-NuiA-TrrnB construct was transformed into competent Escherichia coli TOP10 cells. Colony PCR was used to screen for colonies with the correct insert size. The expected amplicon size for PcopM-NuiA-TrrnB was 1116 bp. Although the negative control and potentially other samples were contaminated, taking that into account all five screened colonies seemed to give the correct band All five screened colonies gave the correct band (Figure 1).

 

Figure 1. Colony PCR of Level 1 NuiA constructs.

Two colonies with the correct cPCR band were screened by restriction digestion with BbsI (cuts out the insert) and NotI (linearizes the plasmid), and the digested DNA was analysed by gel electrophoresis. After restriction digestion, the DNA band for the insert was of approx. expected size (832 bp), while the band showing the Level 1 acceptor backbone and the band showing the whole linearized plasmid were approx. 1 kb bigger compared to the in silico constructs (Figure 2). This extra 1 kb of DNA of unknown origin in the Level 1 acceptor backbone was expected because we previously detected it when digesting the Level 1 vector on its own.

 

Figure 2. Restriction digestion of Level 1 NuiA constructs.

 

We also sent the L1P1-PcopM-NuiA-TrrnB construct to Edinburgh Genome Foundry for sequencing by Oxford Nanopore technology and analysis. The sequencing analysis report showed that most reads of the L1P1-PcopM-NuiA-TrrnB construct (barcode25 / lv1p1_Pcop_NuiA) were of the approx. expected size. The ‘coverage plot’ showed that the entire plasmid was observed, and no large insertions were detected, which confirmed that the Level 1 acceptor backbone did not actually contain the 1 kb insertion observed after restriction digestion, suggesting it was an experimental error. From comparison with reference sequence (Figure 3), some ‘True’ mutations were detected in the backbone of the construct, but not in the insert region.

 

Figure 3. The table lists mutations detected in the L1P1-PcopM-NuiA-TrrnB construct. Those highlighted in red are ‘True’ mutations, whereas those not highlighted are sequences known to be challenging to accurately sequence with nanopores (usually secondary structures / repetitive sequences). In this table, DP is how many reads cover this part of the sequence, RO is how many of those reads contain the expected sequence, AO is how many contain an alternate allele.

 

References

Gale, G.A., Osorio, A.A.S., Puzorjov, A., Wang, B. and McCormick, A.J., 2019. Genetic modification of cyanobacteria by conjugation using the CyanoGate modular cloning toolkit. JoVE (Journal of Visualized Experiments), (152), p.e60451.

Vasudevan, R., Gale, G.A., Schiavon, A.A., Puzorjov, A., Malin, J., Gillespie, M.D., Vavitsas, K., Zulkower, V., Wang, B., Howe, C.J. and Lea-Smith, D.J., 2019. CyanoGate: a modular cloning suite for engineering cyanobacteria based on the plant MoClo syntax. Plant Physiology, 180(1), pp.39-55.